KR102121602B1 - Crushed Stem Cell Extract(Shelled Stem Cell) Manufacturing Method Using Mass Culture Medium Composition Method and Constituent 3-low Extracting Method and A Treating Composition for Anti-Inflammatory and A Treating Composition for Cell Regeneration - Google Patents
Crushed Stem Cell Extract(Shelled Stem Cell) Manufacturing Method Using Mass Culture Medium Composition Method and Constituent 3-low Extracting Method and A Treating Composition for Anti-Inflammatory and A Treating Composition for Cell Regeneration Download PDFInfo
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Abstract
본 발명은 세포배양용 배지 조성물 제조방법 및 세포배양용 배지 조성물 제조방법과 줄기세포 유효성분 3저 추출법을 이용한 줄기세포파쇄추출물 제조방법에 관한 것으로 한다. 세포 배양용 배지 조성물은, 기본배지와, 히알루론산과, 첨가조성물을 포함하여 구성되는 것을 특징으로 한다.
본 발명은 줄기세포의 유효성분을 추출할 때 저온, 저압, 저장이라는 3저 환경에서 줄기세포의 파쇄가 이루어지기 때문에 줄기세포의 유효성분이 손상되는 것을 방지할 수 있는 이점이 있는 것을 특징으로 한다.
본 발명의 다른 실시예인 줄기세포 대량배양용 배지 조성법과 줄기세포 유효성분 3저 추출법을 이용한 줄기세포파쇄추출물 제조방법의 구성은, 줄기세포를 추출하는 제 1단계와, 배지조성물의 배지에 상기 추출된 줄기세포를 배양하는 제 2단계와, 상기 줄기세포를 계대배양하는 제 3단계와, 상기 배양된 줄기세포에서 세포를 획득하는 제 4단계와, 상기 획득된 세포를 파쇄하는 제 5단계와, 상기 파쇄된 물질을 여과하는 제 6단계를 포함하여 구성되는 것을 특징으로 한다. The present invention relates to a method for preparing a medium composition for cell culture, a method for preparing a medium composition for cell culture, and a method for preparing a stem cell crushing extract using three low extraction methods for stem cell active ingredients. The cell culture medium composition is characterized by comprising a basic medium, hyaluronic acid, and an additive composition.
The present invention is characterized in that when extracting the active ingredient of the stem cells, the stem cells are crushed in three low environments such as low temperature, low pressure, and storage, thereby preventing the active ingredient of the stem cells from being damaged.
In another embodiment of the present invention, the composition of a method for preparing a medium for bulk culture of stem cells and a method for preparing a stem cell crushing extract using a three-way extraction method of stem cell active ingredients includes: a first step of extracting stem cells, and extracting the medium into a medium of the medium composition A second step of culturing the stem cells, a third step of subculturing the stem cells, a fourth step of obtaining cells from the cultured stem cells, and a fifth step of crushing the obtained cells, And a sixth step of filtering the crushed material.
Description
본 발명은 세포배양용 배지 조성물 제조방법에 관한 것으로, 더욱 상세하게는 줄기세포를 대량으로 배양하여 수득률을 높일 수 있고, 줄기세포 내의 유효성분 수득률을 높일 수 있는 세포배양용 배지 조성물 제조방법 및 세포배양용 배지 조성물 제조방법과 줄기세포 유효성분 3저 추출법을 이용한 줄기세포파쇄추출물 제조방법, 이를 이용한 항염증 치료용 조성물 및 세포재생 치료용 조성물에 관한 것이다. The present invention relates to a method for preparing a medium composition for cell culture, and more specifically, a method for producing a cell culture medium composition and cells capable of culturing a large number of stem cells to increase the yield, and to increase the yield of active ingredients in the stem cells. It relates to a method for preparing a medium composition for culture, a method for preparing a stem cell crushing extract using a three-cell stem cell active ingredient extraction method, an anti-inflammatory composition using the same, and a composition for treating cell regeneration.
줄기세포(stem cell)는 여러 종류의 신체 조직으로 분화할 수 있는 능력을 가진 미분화 세포로, 다양한 조직 세포로 분화할 수 있는 능력 때문에 이를 이용한 연구가 많이 행해지고 있다. 줄기세포 중 성체 줄기세포는 지방, 골수, 재대혈 또는 태반 등과 같은 부위에서 쉽게 얻을 수 있으며, 배아 줄기세포에 비해 윤리적 문제가 적고, 사용자 본인의 세포를 이용하면 면역거부반응도 적기 때문에 이를 이용한 연구가 많이 이루어지고 있다.Stem cells are undifferentiated cells with the ability to differentiate into various types of body tissues, and many studies have been conducted using them because of the ability to differentiate into various tissue cells. Among stem cells, adult stem cells can be easily obtained from areas such as fat, bone marrow, large blood, or placenta, and have fewer ethical problems compared to embryonic stem cells. Much is being done.
성체 줄기세포를 바로 주입하는 치료는 면역거부반응 등의 이유로 자가치료에 한정된다. 이 자가치료의 한계를 뛰어넘기 위해서는 줄기세포를 파쇄하여 면역거부반응을 일으키는 면역원성이 붙어 있는 세포막을 제거하는 것이 하나의 방법이 될 수 있다. 본원발명의 발명자는 상기 줄기세포파쇄추출물의 개념을 처음으로 정립하고 그 원천 특허를 출원한 바 있다. 그 원천 특허의 장점은 면역거부반응을 일으키는 면역원성이 붙어 있는 세포막을 제거함으로써 누구나 사용이 가능한 범용성이 있기 때문에 대량배양을 할 이유가 성립되고, 그 결과 줄기세포의 대중화와 산업화를 이룰 수 있다. Treatment in which adult stem cells are directly injected is limited to self-treatment for reasons such as an immune rejection reaction. To overcome the limitations of this self-treatment, one way is to remove the cell membrane with immunogenicity that causes an immune rejection by crushing stem cells. The inventor of the present invention first established the concept of the stem cell crushing extract and applied for the original patent. The advantage of the original patent is that the reason for mass cultivation is established because it has versatility that anyone can use by removing the cell membrane attached to the immunogenicity that causes an immune rejection reaction. As a result, it is possible to achieve popularization and industrialization of stem cells.
그래서, 줄기세포의 대중화와 산업화를 이루기 위해서 줄기세포의 대량배양에 유용한 방법과 그 유효성분을 추출하는 방법의 지속적인 연구 및 개발이 요구되고 있다. Therefore, in order to achieve the popularization and industrialization of stem cells, continuous research and development of a method useful for mass culturing stem cells and a method for extracting the active ingredient thereof is required.
상술한 문제점을 해결하기 위한 것으로, 본 발명의 목적은 줄기세포를 대량으로 배양하여, 세포의 수득을 높이는 줄기세포 대량배양용 배지 조성법과, 줄기세포 유효성분 수집율을 높일 수 있는 줄기세포 유효성분 3저 추출법을 이용한 줄기세포파쇄추출물 제조방법을 제공하는 것이다. In order to solve the above-mentioned problems, the object of the present invention is to cultivate stem cells in a large amount, a method for forming a medium for mass culture of stem cells to increase the yield of cells, and stem cell active ingredients that can increase the collection rate of stem cell active ingredients. 3 to provide a method for producing stem cell crushed extract using a low extraction method.
상술한 목적을 달성하기 위한 것으로, 본 발명인 세포 배양용 배지 조성물 제조방법은, 기본배지와, 히알루론산과, 첨가조성물을 포함하여 구성되는 것을 특징으로 한다. To achieve the above object, the method for preparing a cell culture medium composition according to the present invention is characterized in that it comprises a basic medium, hyaluronic acid, and an additive composition.
상기 첨가조성물은, 글리신, 히스티딘, 이소류신, 메티오닌, 페닐알라닌, 프롤린, 히드록시프롤린, 세린, 트레오닌, 트립토판, 티로신, 발린, bFGF, EGF, VEGF, KGF, HGF, TGF, 비타민C, 비타민B1, 비타민B12, 비타민E, 셀레늄, 트랜스페린을 포함하여 구성되는 것을 특징으로 한다. The additive composition, glycine, histidine, isoleucine, methionine, phenylalanine, proline, hydroxyproline, serine, threonine, tryptophan, tyrosine, valine, bFGF, EGF, VEGF, KGF, HGF, TGF, vitamin C, vitamin B1, vitamin It is characterized by being composed of B12, vitamin E, selenium, and transferrin.
상기 기본배지는, DMEM(Dulbecco's Modified Eagle's Medium), MEM(Minimal Essential Medium), BME(Basal Medium Eagle), RPMI 1640, F-10, F12, DMEM-F12, α-MEM(α-Minimal Essential Medium), G-MEM(Glasgow's Minimal Essential Medium), IMDM(Iscove's Modified Dulbecco's Medium), MacCoy's 5A 배지, AmnioMax, AminoMaxⅡ complete Medium, Chang's Medium MesemCult-XF Medium 중 어느 하나인 것을 특징으로 한다. The basic medium, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F12, DMEM-F12, α-MEM (α-Minimal Essential Medium) , G-MEM (Glasgow's Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), MacCoy's 5A medium, AmnioMax, AminoMax II complete Medium, Chang's Medium MesemCult-XF Medium.
상기 첨가조성물은 상기 배지조성물에 대하여 히알루론산은 10㎍/㎖의 농도로 포함되고, 상기 글리신은 1ng/㎖의 농도로 포함되고, 상기 히스티딘은 1ng/㎖의 농도로 포함되고, 상기 이소류신은 1ng/㎖의 농도로 포함되고, 상기 메티오닌은 1ng/㎖의 농도로 포함되고, 상기 페닐알라닌은 1ng/㎖의 농도로 포함되고, 상기 프롤린은 10ng/㎖의 농도로 포함되고, 상기 히드록시프롤린은 5ng/㎖의 농도로 포함되고, 상기 세린은 1ng/㎖의 농도로 포함되고, 상기 트레오닌은 1ng/㎖의 농도로 포함되고, 상기 트립토판은 1ng/㎖의 농도로 포함되고, 상기 티로신은 1ng/㎖의 농도로 포함되고, 상기 발린은 2ng/㎖의 농도로 포함되고, 상기 bFGF는 9㎍/㎖의 농도로 포함되고, 상기 EGF는 1.5㎍/㎖의 농도로 포함되고, 상기 VEGF는 1㎍/㎖의 농도로 포함되고, 상기 KGF는 1.2㎍/㎖의 농도로 포함되고, 상기 HGF는 0.5㎍/㎖의 농도로 포함되고, 상기 TGF는 0.5㎍/㎖의 농도로 포함되고, 상기 비타민C는2㎍/㎖의 농도로 포함되고, 상기 비타민B1는 0.5㎍/㎖의 농도로 포함되고, 상기 비타민B12는 3㎍/㎖의 농도로 포함되고, 상기 비타민E는 500㎍/㎖의 농도로 포함되고, 상기 셀레늄은 1.8㎍/㎖의 농도로 포함되고, 상기 트랜스페린은 12㎍/㎖의 농도로 포함되는 것을 특징으로 한다. The additive composition contains hyaluronic acid at a concentration of 10 μg/ml, the glycine is contained at a concentration of 1 ng/ml, the histidine is contained at a concentration of 1 ng/ml, and the isoleucine is 1 ng with respect to the medium composition. /Ml, the methionine is contained at a concentration of 1ng/ml, the phenylalanine is contained at a concentration of 1ng/ml, the proline is contained at a concentration of 10ng/ml, and the hydroxyproline is 5ng /Ml, the serine is contained at a concentration of 1ng/ml, the threonine is contained at a concentration of 1ng/ml, the tryptophan is contained at a concentration of 1ng/ml, and the tyrosine is 1ng/ml Is contained in the concentration, the valine is contained in a concentration of 2ng / ㎖, the bFGF is contained in a concentration of 9㎍ / ㎖, the EGF is contained in a concentration of 1.5㎍ / ㎖, the VEGF is 1㎍ / It is contained in a concentration of ㎖, the KGF is contained in a concentration of 1.2㎍ / ㎖, the HGF is contained in a concentration of 0.5㎍ / ㎖, the TGF is contained in a concentration of 0.5㎍ / ㎖, the vitamin C is 2 ㎍ / ㎖ concentration, the vitamin B1 is contained in a concentration of 0.5 ㎍ / ㎖, the vitamin B12 is contained in a concentration of 3 ㎍ / ㎖, the vitamin E is included in a concentration of 500 ㎍ / ㎖ It is characterized in that the selenium is contained in a concentration of 1.8 ㎍ / ㎖, the transferrin is included in a concentration of 12 ㎍ / ㎖.
그리고, 줄기세포 대량배양용 배지 조성법과 줄기세포 유효성분 3저 추출법을 이용한 줄기세포파쇄추출물 제조방법은, 줄기세포를 추출하는 제 1단계와, 상술한 배지조성물의 배지에 상기 추출된 줄기세포를 배양하는 제 2단계와, 상기 줄기세포를 계대배양하는 제 3단계와, 상기 배양된 줄기세포에서 세포를 획득하는 제 4단계와, 상기 획득된 세포를 파쇄하는 제 5단계와, 상기 파쇄된 물질을 여과하는 제 6단계와, 상시 6단계 여과된 물질을 동결건조하여 보관 하거나 활용하는 7단계를 포함하여 구성되는 것을 특징으로 한다. In addition, the method for preparing a stem cell crushed extract using the medium composition method for mass culture of stem cells and the extraction method for stem cell
상기 제 5단계에서, 내부에 공간이 형성되는 외부와 내부가 차단되는 몸체부(10)와, 상기 몸체부(10) 내부에 마련되고 상방이 개구되는 제 1용기부(12)와, 상기 제 1용기부(12)에 대해 상대적으로 작은 크기로 형성되며, 상기 제 1용기부(12) 내에 마련되며 상방이 개구되어 내부에 줄기세포가 투입되는 제 2용기부(14)와, 상기 제 2용기부(14) 내부의 줄기세포를 파쇄하는 파쇄부(16)와, 상기 몸체부(10) 내부로 공기의 유동을 단속하는 밸브(18)와, 상기 몸체부(10) 내부에서 공기를 흡입하는 펌프(20)를 포함하는 줄기세포 유효성분 추출장치를 이용하고, 상기 줄기세포는 저장성 용액에 투입후 상기 제 2용기부에 투입하는 것을 특징으로 한다. In the fifth step, the
본 발명의 다른 실시예인 줄기세포파쇄추출물을 이용한 항염증 치료용 조성물인 것을 특징으로 한다. Another embodiment of the present invention is characterized in that the composition for the treatment of anti-inflammatory using stem cell crushed extract.
본 발명의 또 다른 실시예인 줄기세포파쇄추출물을 이용한 세포재생 치료용 조성물인 것을 특징으로 한다. Another embodiment of the present invention is characterized in that it is a composition for cell regeneration treatment using a stem cell crushing extract.
본 발명의 또 다른 실시예인 줄기세포파쇄추출물을 이용한 항관절염 치료용 조성물인 것을 특징으로 한다. Another embodiment of the present invention is characterized in that the composition for the treatment of anti-arthritis using a stem cell crushing extract.
본 발명에 의한 세포배양용 배지 조성물 제조방법 및 세포배양용 배지 조성물 제조방법과 줄기세포 유효성분 3저 추출법을 이용한 줄기세포파쇄추출물 제조방법의 효과는 다음과 같다. The effects of the method for preparing a medium composition for cell culture and the method for preparing a medium composition for cell culture and the method for preparing a stem cell crushing extract using the stem cell
혈청을 사용하지 않아 안정하며, 스캐폴드를 사용하여 배양이 증대되어 줄기세포의 유효인자의 수득률이 높아지는 이점이 있다. It is stable because it does not use serum, and the culture is increased by using a scaffold, thereby increasing the yield of effective factors of stem cells.
줄기세포의 유효성분을 추출할 때 저온, 저압, 저장이라는 3저 환경에서 줄기세포의 파쇄가 이루어지기 때문에 줄기세포의 유효성분이 손상되는 것을 방지할 수 있는 이점이 있다. When extracting the active ingredient of the stem cells, since the crushing of the stem cells is performed in the three low environment of low temperature, low pressure, and storage, there is an advantage that the active ingredient of the stem cells can be prevented from being damaged.
그리고, 상기 두 가지 방법을 이용한 줄기세포파쇄추출물은 면역거부반응을 일으키는 면역원성이 붙은 세포막이 완전히 제거되어 누구나 사용이 가능한 범용성이 있어서 줄기세포의 대중화와 산업화를 이룰 수 있는 이점이 있다. In addition, the stem cell crushing extract using the above two methods has the advantage of being able to achieve the popularization and industrialization of stem cells because the cell membrane with the immunogenicity causing an immune rejection reaction is completely removed and versatile for anyone to use.
도 1은 본 발명에 의한 줄기세포 유효성분 3저 추출장치를 보인 도면.
도 2는 본 발명에 의한 줄기세포파쇄추출물의 항 관절염 효과를 확인하기 위하여 연골세포의 특정마커인 CD44를 이용하여 연골세포임을 확인하는 도면.
도 3은 연골세포에 줄기세포파쇄성분추출물을 농도별 처리하여 24시간 배양, 세포생존율을 측정하여 물질의 세포에 대한 독성을 확인하는 도면.
도 4는 연골세포에 IL-1α를 처리해 염증을 일으킨 후, 염증을 일으킨 연골세포에 줄기세포파쇄추출물을 농도별 처리하여 염증매개 인자인 NO생성 억제를 확인한 도면.
도 5는 IL-1α 처리로 유도된 연골세포에 줄기세포파쇄추출물을 처리한 후 유도된 NO의 생성 억제 효과는 iNOS와 COX-2의 발현 억제와 연관을 확인한 도면.
도 6은 줄기세포파쇄추출물을 처리한 연골세포는 IL-1α에 의한 NF-κB 단백질이 핵내로 이동되는 것을 억제하는 것을 확인한 도면.
도 7은 IL-1α 에 의해 염증이 발생한 연골세포에 줄기세포파쇄추출물을 처리하여 iMAPKs 활성을 확인한 도면.
도 8은 유전학적 발현을 통해 본 줄기세포추출물의 항염증 효과 확인하는 도면.
도 9는 연골 형성 발달에 관여하는 sox-9의 유전자를 통해 연골형성 효과를 확인하는 도면.
도 10은 본 발명에 의한 줄기세포파쇄추출물을 농도별 처리한 결과 유의한 세포 독성의 발현 유무를 보인 도면.
도 11은 본 발명에 의한 줄기세포파쇄추출물을 LPS와 동시에 처리한 결과 세포 독성의 발현유무를 보인 도면.
도 12는 본 발명에 의한 줄기세포파쇄추출물을 LPS와 처리한 결과 세포 형태의 변화를 보인 도면.
도 13은 본 발명에 의한 줄기세포파쇄추출물을 LPS와 처리한 결과 산화질소 농도의 변화를 보인 도면.
도 14는 본 발명에 의한 줄기세포파쇄추출물을 LPS와 처리한 결과 iNOS 및 COX-2 단백질 발현 변화를 보인 도면.
도 15는 본 발명에 의한 줄기세포파쇄추출물을 LPS와 처리한 결과 TNFα의 변화를 보인 도면
도 16은 본 발명에 의한 줄기세포파쇄추출물을 LPS와 처리한 결과 IL-1β의 변화를 보인 도면.1 is a view showing an extraction device for three active stem cells according to the present invention.
2 is to confirm the anti-arthritis effect of stem cell crushing extract according to the present invention Drawing to confirm chondrocytes using CD44, a specific marker of chondrocytes.
3 is a view to check the toxicity of the substance to the cells by measuring the cell viability for 24 hours by processing the stem cell crushing component extract in chondrocytes by concentration.
FIG. 4 is a diagram showing the inhibition of NO production as an inflammation mediator by treating IL-1α on chondrocytes to cause inflammation, and then treating the inflammatory chondrocytes with stem cell crushing extract according to concentration.
5 is a view confirming the effect of inhibiting the production of induced NO after inhibiting the expression of iNOS and COX-2 after treating the stem cell crushing extract on chondrocytes induced by IL-1α treatment.
6 is a view confirming that chondrocytes treated with the stem cell crushing extract inhibit the migration of NF-κB protein by IL-1α into the nucleus.
7 is treated with stem cell crushing extract to chondrocytes inflamed by IL-1α Drawing confirming iMAPKs activity.
8 is a diagram confirming the anti-inflammatory effect of the stem cell extract seen through genetic expression.
9 is a view for confirming the effect of cartilage formation through the gene of sox-9 involved in cartilage formation development.
10 is a view showing the presence or absence of significant cytotoxicity as a result of treating the stem cell crushing extract according to the present invention by concentration.
11 is a view showing the presence or absence of cytotoxicity as a result of processing the stem cell crushing extract according to the present invention simultaneously with LPS.
12 is a view showing a change in cell morphology as a result of treating the stem cell crushing extract according to the present invention with LPS.
13 is a view showing a change in nitrogen oxide concentration as a result of treating the stem cell crushing extract according to the present invention with LPS.
14 is a view showing the iNOS and COX-2 protein expression changes as a result of treating the stem cell crushing extract according to the present invention with LPS.
15 is a view showing the change in TNFα as a result of treating the stem cell crushing extract according to the present invention with LPS
16 is a view showing a change in IL-1β as a result of treating the stem cell crushing extract according to the present invention with LPS.
이하, 본 발명에 의한 세포배양용 배지 조성물 제조방법 및 세포배양용 배지 조성물 제조방법과 줄기세포 유효성분 3저 추출법을 이용한 줄기세포파쇄추출물 제조방법의 바람직한 실시예가 첨부된 도면을 참고하여 상세하게 설명한다. Hereinafter, with reference to the accompanying drawings, preferred embodiments of the method for preparing a cell culture medium composition and a method for preparing a cell culture medium composition and a method for producing stem cell crushing extract using the stem cell
먼저, 본 발명인 줄기세포 유효성분 추출장치는 도 1에 도시된 바와 같이, 내부에 공간이 형성되는 외부와 내부가 차단되는 몸체부(10)와, 상기 몸체부(10) 내부에 마련되고 상방이 개구되는 제 1용기부(12)와, 상기 제 1용기부(12)에 대해 상대적으로 작은 크기로 형성되며, 상기 제 1용기부(12) 내에 마련되며 상방이 개구되어 내부에 줄기세포가 투입되는 제 2용기부(14)와, 상기 제 2용기부(14) 내부의 줄기세포를 파쇄하는 파쇄부(16)와, 상기 몸체부(10) 내부로 공기의 유동을 단속하는 밸브(18)와, 상기 몸체부(10) 내부에서 공기를 흡입하는 펌프(20)를 포함하여 구성될 수 있다. First, as shown in FIG. 1, the stem cell active ingredient extracting device according to the present invention is provided with a
먼저, 본 발명인 추출장치에는 몸체부(10)가 마련된다. 상기 몸체부(10)는 도 1에 도시된 바와 같이, 내부에 밀폐된 공간이 마련되어 외부와 내부가 차단되도록 한다. First, the
상기 몸체부(10) 내부에는, 제 1용기부(12)가 마련된다. 상기 제 1용기부(12)는 내부에 공간이 마련되고 상부가 개방되도록 형성된다. 상기 제 1용기부(12)에는 얼음이 적재되어 아래에서 설명될 제 2용기부(14) 내부의 줄기세포의 온도를 낮게 만드는 역할을 한다. Inside the
상기 제 1용기부(12) 내부에는 제 2용기부(14)가 마련된다. 상기 제 2용기부(14)는 상기 제 1용기부(12)와 마찬가지로 내부에 공간이 마련되고 상부가 개방되도록 형성된다. 특히, 상기 제 2용기부(14)는 상기 제 1용기부(12) 보다 상대적으로 작은 크기로 형성되며, 상기 제 2용기부(14)는 상기 제 1용기부(12) 내부에 적재될 수 있다. 상기 제 2용기부(14)에는 줄기세포가 적재될 수 있다. A
그리고, 본 발명에는 파쇄부(16)가 더 마련될 수 있다. 상기 파쇄부(16)는 상기 제 2용기부(14) 내부에 적재되는 줄기세포를 초음파를 이용하여 파쇄하는 역할을 한다. And, in the present invention, a crushing
또한, 본 발명에는 외부와 공기가 유동되는 유로가 마련되며, 상기 유로를 통한 공기의 흐름을 선택적으로 차단하는 밸브(18)가 마련된다. 상기 유로의 타단에는 펌프(20)가 마련되어 상기 몸체부(10) 내부의 공기를 외부로 배출시켜 상기 몸체부(10) 내부를 진공 상태로 만들 수 있다.In addition, the present invention is provided with a flow path through which the outside and air flow, and a
또한, 상기 제 2용기부에 투입되는 줄기세포는 저장액에 넣어 상기 줄기세포가 팽창되도록 할 수 있다. 즉, 상기 줄기세포를 증류수 같은 저장액에 넣어 상기 줄기세포가 삼투압에 의하여 팽창되도록 하여 작은 충격으로 파쇄되도록 할 수 있다. In addition, the stem cells introduced into the second container portion may be placed in a storage solution to expand the stem cells. That is, the stem cells can be put into a storage solution such as distilled water to cause the stem cells to expand by osmotic pressure so that they can be crushed with a small impact.
그리고, 본 발명에 의한 줄기세포 대량배양용 배지조성물 및 의 배지조성방법에 대해 상세하게 설명한다. And, the medium composition for mass culture of stem cells according to the present invention and the medium composition method of will be described in detail.
본 발명에서는 새로운 배지를 제조하여 세포수득량을 높이고, 세포를 파쇄하였을 때 유효성분을 더욱 많이 수득할 수 있도록 한다. In the present invention, by preparing a new medium to increase the cell yield, it is possible to obtain more active ingredients when the cells are crushed.
상기 배지는 기본 배지는 동물 세포의 배양에 통상적으로 사용되는 공지의 배지들로서, 예컨대 DMEM(Dulbecco's Modified Eagle's Medium), MEM(Minimal Essential Medium), BME(Basal Medium Eagle), RPMI 1640, F-10, F12, DMEM-F12, α-MEM(α-Minimal Essential Medium), G-MEM(Glasgow's Minimal Essential Medium), IMDM(Iscove's Modified Dulbecco's Medium), MacCoy's 5A 배지, AmnioMax, AminoMaxⅡ complete Medium, Chang's Medium MesemCult-XF Medium 등일 수 있으나, 특별히 이에 한정되지 아니한다.The medium is a known medium that is commonly used for culturing animal cells, for example, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F12, DMEM-F12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), MacCoy's 5A medium, AmnioMax, AminoMaxII complete Medium, Chang's Medium MesemCult-XF Medium, etc., but is not particularly limited thereto.
그리고, 상기 배지에 첨가조성물을 추가한다. 상기 첨가조성물은 히알루론산, 글리신, 히스티딘, 이소류신, 메티오닌, 페닐알라닌, 프롤린, 히드록시프롤린, 세린, 트레오닌, 트립토판, 티로신, 발린, bFGF, EGF, VEGF, KGF, HGF, TGF, 비타민C, 비타민B1, 비타민B12, 비타민E, 셀레늄, 트랜스페린을 포함하여 구성될 수 있다. Then, an additive composition is added to the medium. The additive composition is hyaluronic acid, glycine, histidine, isoleucine, methionine, phenylalanine, proline, hydroxyproline, serine, threonine, tryptophan, tyrosine, valine, bFGF, EGF, VEGF, KGF, HGF, TGF, vitamin C, vitamin B1 , Vitamin B12, vitamin E, selenium, transferrin.
먼저, 상기 첨가조성물 중 히알루론산이 포함된다. 상기 히알루론산은 세포배양시 스캐폴드 역할을 하는 것으로, 상기 배지조성물에 대하여 10㎍/㎖의 농도로 포함될 수 있다. First, hyaluronic acid is included in the addition composition. The hyaluronic acid serves as a scaffold during cell culture, and may be included in a concentration of 10 μg/ml with respect to the medium composition.
그리고, 상기 첨가조성물에는 글리신이 포함된다. 상기 글리신은 아미노산으로 세포의 성장에 영양성분 역할을 하는 것으로, 상기 배지조성물에 대하여 1ng/㎖의 농도로 포함될 수 있다. And, the additive composition contains glycine. The glycine is an amino acid that serves as a nutrient for cell growth, and may be included in a concentration of 1 ng/ml with respect to the medium composition.
상기 첨가조성물에는 히스티딘이 포함된다 상기 히스티딘은 아미노산으로 세포의 성장에 영양성분 역할을 하는 것으로, 상기 배지조성물에 대하여 1ng/㎖의 농도로 포함될 수 있다.The addition composition includes histidine. The histidine is an amino acid, which serves as a nutrient for cell growth, and may be included in a concentration of 1 ng/ml with respect to the medium composition.
상기 첨가조성물에는 이소류신이 포함된다 상기 이소류신은 아미노산으로 세포의 성장에 영양성분 역할을 하는 것으로, 상기 배지조성물에 대하여 1ng/㎖의 농도로 포함될 수 있다.The additive composition includes isoleucine. Isoleucine is an amino acid that serves as a nutrient for cell growth, and may be included in a concentration of 1 ng/ml with respect to the medium composition.
상기 첨가조성물에는 메티오닌이 포함된다 상기 메티오닌은 아미노산으로 세포의 성장에 영양성분 역할을 하는 것으로, 상기 배지조성물에 대하여 1ng/㎖의 농도로 포함될 수 있다.Methionine is included in the additive composition. The methionine is an amino acid that serves as a nutrient for cell growth, and may be included in a concentration of 1 ng/ml with respect to the medium composition.
상기 첨가조성물에는 페닐알라닌이 포함된다 상기 페닐알라닌은 아미노산으로 세포의 성장에 영양성분 역할을 하는 것으로, 상기 배지조성물에 대하여 1ng/㎖의 농도로 포함될 수 있다.The additive composition includes phenylalanine. The phenylalanine is an amino acid that serves as a nutrient for cell growth, and may be included in a concentration of 1 ng/ml with respect to the medium composition.
상기 첨가조성물에는 프롤린 포함된다 상기 프롤린은 아미노산으로 세포의 성장에 영양성분 역할을 하는 것으로, 상기 배지조성물에 대하여 10ng/㎖의 농도로 포함될 수 있다.The additive composition includes proline. The proline is an amino acid, which serves as a nutrient for cell growth, and may be included in a concentration of 10 ng/ml with respect to the medium composition.
상기 첨가조성물에는 히드록시프롤린이 포함된다 상기 히드록시프롤린은 아미노산으로 세포의 성장에 영양성분 역할을 하는 것으로, 상기 배지조성물에 대하여 5ng/㎖의 농도로 포함될 수 있다.The additive composition includes hydroxyproline. The hydroxyproline is an amino acid that serves as a nutrient for cell growth, and may be included in a concentration of 5 ng/ml with respect to the medium composition.
상기 첨가조성물에는 세린이 포함된다 상기 세린은 아미노산으로 세포의 성장에 영양성분 역할을 하는 것으로, 상기 배지조성물에 대하여 1ng/㎖의 농도로 포함될 수 있다.Serine is included in the additive composition. Serine is an amino acid, which serves as a nutrient for cell growth, and may be included in a concentration of 1 ng/ml with respect to the medium composition.
상기 첨가조성물에는 트레오닌이 포함된다 상기 트레오닌은 아미노산으로 세포의 성장에 영양성분 역할을 하는 것으로, 상기 배지조성물에 대하여 1ng/㎖의 농도로 포함될 수 있다.The additive composition includes threonine. The threonine is an amino acid that serves as a nutrient for cell growth, and may be included in a concentration of 1 ng/ml with respect to the medium composition.
상기 첨가조성물에는 트립토판이 포함된다 상기 트립토판은 아미노산으로 세포의 성장에 영양성분 역할을 하는 것으로, 상기 배지조성물에 대하여 1ng/㎖의 농도로 포함될 수 있다.The additive composition includes tryptophan. The tryptophan is an amino acid that serves as a nutrient for cell growth, and may be included in a concentration of 1 ng/ml with respect to the medium composition.
상기 첨가조성물에는 티로신이 포함된다 상기 티로신은 아미노산으로 세포의 성장에 영양성분 역할을 하는 것으로, 상기 배지조성물에 대하여 1ng/㎖의 농도로 포함될 수 있다.The additive composition includes tyrosine. The tyrosine is an amino acid, which serves as a nutrient for cell growth, and may be included in a concentration of 1 ng/ml with respect to the medium composition.
상기 첨가조성물에는 발린이 포함된다 상기 발린은 아미노산으로 세포의 성장에 영양성분 역할을 하는 것으로, 상기 배지조성물에 대하여 2ng/㎖의 농도로 포함될 수 있다.The addition composition contains valine. The valine is an amino acid that serves as a nutrient for cell growth, and may be included in a concentration of 2 ng/ml with respect to the medium composition.
그리고, 상기 첨가조성물에는 bFGF가 포함된다. 상기 bFGF는 배양되는 세포의 성장을 돕는 것으로, 상기 배지조성물에 대하여 9㎍/㎖의 농도로 포함될 수 있다. And, the addition composition includes bFGF. The bFGF is to help the growth of the cultured cells, it may be included in a concentration of 9㎍ / ㎖ with respect to the medium composition.
상기 첨가조성물에는 EGF가 포함된다. 상기 EGF는 배양되는 세포의 성장을 돕는 것으로, 상기 배지조성물에 대하여 1.5㎍/㎖의 농도로 포함될 수 있다. EGF is included in the additive composition. The EGF is to help the growth of the cultured cells, it may be included in a concentration of 1.5㎍ / ㎖ with respect to the medium composition.
상기 첨가조성물에는 VEGF가 포함된다. 상기 VEGF는 배양되는 세포의 성장을 돕는 것으로, 상기 배지조성물에 대하여 1㎍/㎖의 농도로 포함될 수 있다. The addition composition includes VEGF. The VEGF is to help the growth of the cultured cells, it may be included in a concentration of 1㎍ / ㎖ with respect to the medium composition.
상기 첨가조성물에는 KGF가 포함된다. 상기 KGF는 배양되는 세포의 성장을 돕는 것으로, 상기 배지조성물에 대하여 1.2㎍/㎖의 농도로 포함될 수 있다. The additive composition includes KGF. The KGF is to help the growth of the cultured cells, it may be included in a concentration of 1.2㎍ / ㎖ with respect to the medium composition.
상기 첨가조성물에는 HGF가 포함된다. 상기 HGF는 배양되는 세포의 성장을 돕는 것으로, 상기 배지조성물에 대하여 0.5㎍/㎖의 농도로 포함될 수 있다. The additive composition includes HGF. The HGF is to help the growth of the cultured cells, it may be included in a concentration of 0.5㎍ / ㎖ with respect to the medium composition.
상기 첨가조성물에는 TGF가 포함된다. 상기 TGF는 배양되는 세포의 성장을 돕는 것으로, 상기 배지조성물에 대하여 0.5㎍/㎖의 농도로 포함될 수 있다. TGF is included in the additive composition. The TGF is to help the growth of the cultured cells, it may be included in a concentration of 0.5㎍ / ㎖ with respect to the medium composition.
그리고, 상기 첨가조성물에는 비타민C가 포함된다. 상기 비타민C는 배양되는 세포의 성장을 돕는 것으로, 상기 배지조성물에 대하여 2㎍/㎖의 농도로 포함될 수 있다. In addition, the additive composition includes vitamin C. The vitamin C is to help the growth of the cultured cells, it may be included in a concentration of 2㎍ / ㎖ with respect to the medium composition.
상기 첨가조성물에는 비타민B1가 포함된다. 상기 비타민B1는 배양되는 세포의 성장을 돕는 것으로, 상기 배지조성물에 대하여 0.5㎍/㎖의 농도로 포함될 수 있다. The additive composition includes vitamin B1. The vitamin B1 is to help the growth of the cultured cells, it may be included in a concentration of 0.5㎍ / ㎖ with respect to the medium composition.
상기 첨가조성물에는 비타민B12가 포함된다. 상기 비타민B12는 배양되는 세포의 성장을 돕는 것으로, 상기 배지조성물에 대하여 3㎍/㎖의 농도로 포함될 수 있다. The additive composition includes vitamin B12. The vitamin B12 is to help the growth of cells to be cultured, it may be included in a concentration of 3㎍ / ㎖ with respect to the medium composition.
상기 첨가조성물에는 비타민E가 포함된다. 상기 비타민E는 배양되는 세포의 성장을 돕는 것으로, 상기 배지조성물에 대하여 500㎍/㎖의 농도로 포함될 수 있다. Vitamin E is included in the additive composition. The vitamin E is to help the growth of the cultured cells, it may be included in a concentration of 500㎍ / ㎖ with respect to the medium composition.
그리고, 상기 첨가조성물에는 셀레늄이 포함된다. 상기 셀레늄은 세포의 활성을 돕는 것으로, 상기 배지조성물에 대하여 1.8㎍/㎖의 농도로 포함될 수 있다. And, the additive composition contains selenium. The selenium is to help the activity of the cell, it may be included in a concentration of 1.8㎍ / ㎖ with respect to the medium composition.
상기 첨가조성물에는 트랜스페린이 포함된다. 상기 트랜스페린은 세포의 활성을 돕는 것으로, 상기 배지조성물에 대하여 12㎍/㎖의 농도로 포함될 수 있다. The added composition includes transferrin. The transferrin is to help the activity of the cell, it may be included in a concentration of 12㎍ / ㎖ with respect to the medium composition.
그리고, 본 발명에 의한 줄기세포 유효성분 추출장치를 이용하여 세포배양용 배지 조성물 제조방법 및 세포배양용 배지 조성물 제조방법과 줄기세포 유효성분 3저 추출법을 이용한 줄기세포파쇄추출물 제조방법에 대해 설명한다. Then, a method for preparing a medium composition for cell culture, a method for preparing a medium composition for cell culture, and a method for preparing a stem cell crushing extract using a low extraction method for stem cell active ingredients using the stem cell active ingredient extracting apparatus according to the present invention will be described. .
제 1단계: 줄기세포를 인체의 지방, 골수, 제대혈 또는 태반 중 어느 하나에서 추출한다. 본원발명에서는 지방흡입술로 지방을 수거한 후 효소처리와 여러번 원심분리를 통하여 정제하여 지방줄기세포를 분리한다. Step 1: The stem cells are extracted from any one of human body fat, bone marrow, umbilical cord blood or placenta. In the present invention, fat is collected by liposuction and then purified through enzymatic treatment and centrifugation several times to separate fat stem cells.
제 2단계: 상기 분리된 줄기세포를 상술한 본원발명의 대량배양용 배지조성물을 사용하여 만든 배지를 이용하여 배양한다. 상기 줄기세포 대량배양용 배지조성물을 이용하게 되면 줄기세포의 성장속도를 높여 줄기세포의 수득율이 높아진다. Step 2: The isolated stem cells are cultured using a medium prepared using the medium composition for mass culture of the present invention described above. When the medium composition for mass culture of stem cells is used, the growth rate of stem cells is increased to increase the yield of stem cells.
제 3단계; 줄기세포가 배양된 배양액을 제거한 다음 줄기세포를 분리하고 세포부유액을 수득하여 계대배양한다. The third step; After removing the culture medium in which the stem cells have been cultured, the stem cells are separated and the cell suspension is obtained and subcultured.
제 4단계: 세포가 충분한 밀도로 자라면 세포를 플레이트에서 분리한다. 줄기세포를 분리한 후 생리식염수 또는 PBS(phosphate buffer saline)를 이용하여 수차례 세척한다. Cell counting 기로 세포수를 측정하여 항상 일정한 농도의 부유액을 만든다. Step 4: When the cells grow to a sufficient density, the cells are separated from the plate. After separating the stem cells, they are washed several times with physiological saline or phosphate buffer saline (PBS). Cell counting is used to measure the number of cells to always produce a constant concentration of suspension.
제 5단계; 줄기세포막의 파쇄가 가능하도록 상등액을 제거하고 증량제를 첨가한다. 초음파를 가하여 줄기세포 유효성분 3저 추출법으로 세포를 파쇄한다. 현미경을 통해 줄기세포가 전부 파쇄된 것이 확인되면 파쇄를 종료한다. 상기 3저 추출법의 과정을 거치면 단시간 내 효과적인 세포막 파쇄를 하여 줄기세포 내부의 성장인자와 세포활성물질 등 유효성분의 대부분이 유지된다.
제 6단계: 저장성 증량제와 줄기세포 파쇄 혼합물에는 줄기세포 내용물인 다양한 성장인자, 세포활성물질과 함께 세포막 잔여물이 존재한다. 그 중 필요하지 않은 세포막 잔여물을 원심분리 및 미세필터를 사용하여 제거한다. 이 때 세포막에 붙어있는 면역거부반응을 일으키는 면역원성이 제거되어 누구나 사용 가능한 줄기세포 유효성분이 추출된다. Step 6: The storage membrane extender and the stem cell crushing mixture contain stem cell contents with various growth factors and cell active substances, and a cell membrane residue. Among them, unneeded cell membrane residues are removed by centrifugation and microfiltering. At this time, the immunogenicity causing an immune rejection reaction attached to the cell membrane is removed, and the stem cell active ingredient usable by anyone is extracted.
제 7단계; 상기를 거쳐 만든 줄기세포 파쇄추출물을 냉동 보관하거나 동결 건조하여 보관하고, 활용한다. Step 7; The stem cell crushed extract made through the above is stored frozen or freeze-dried and stored.
상기 단계를 거쳐 얻은 유효성분들을 줄기세포파쇄추출물이라 하며 일명 쉘드줄기세포(Shelled Stem Cell)라 한다. The active ingredients obtained through the above steps are referred to as stem cell crushing extracts and are also called shelled stem cells.
이하, 본 발명에 의한 첨가조성물에 의한 줄기세포 대량배양용 배지 조성물을 이용하여 줄기세포의 파쇄추출물 유효성분 추출하여 실험한 결과를 나타낸 것이다. Hereinafter, using the medium composition for mass culture of stem cells by the additive composition according to the present invention shows the results of experiments by extracting the active ingredient of the crushed extract of stem cells.
[ 비교예 1 ][Comparative Example 1]
DMEM(Dulbecco's Modified Eagle Medium) 100중량부에 혈청으로 FBS가 11,12중량부 첨가되고, 항생제로 penicillin이 0.6중량부 혼합한 배지에 줄기세포를 배양하고, 이를 상술한 과정에 의하여 성장시키고, 유효인자를 추출하였다. DMEM (Dulbecco's Modified Eagle Medium) was added with 11,12 parts by weight of FBS as serum in 100 parts by weight, and cultured stem cells in a medium mixed with 0.6 parts by weight of penicillin with antibiotics. Factors were extracted.
[ 실시예 2 ][Example 2]
DMEM(Dulbecco's Modified Eagle Medium) 100중량부에 혈청이 포함되지 않고 영양성분들이 함유된 세럼(serum) 20중량부 혼합한 배지에 줄기세포를 배양하고, 이를 상술한 과정을 따라 성장시키고, 유효인자를 추출하였다. DMEM (Dulbecco's Modified Eagle Medium) cultured stem cells in a medium mixed with 20 parts by weight of serum containing no nutrients and containing 100 parts by weight of serum, grown according to the process described above, and effective factors Extracted.
[ 실시예 3 ][Example 3]
DMEM(Dulbecco's Modified Eagle Medium) 100중량부에 표 1에 나타난 본 발명의 첨가조성물을 5중량부 혼합한 배지에 줄기세포를 배양하고, 이를 상술한 과정을 따라 성장시키고, 유효인자를 추출하였다.Stem cells were cultured in a medium mixed with 5 parts by weight of the additive composition of the present invention shown in Table 1 in 100 parts by weight of DMEM (Dulbecco's Modified Eagle Medium), grown according to the procedure described above, and extracted with effective factors.
[ 실시예 4 ][Example 4]
DMEM(Dulbecco's Modified Eagle Medium) 100중량부에 표 1에 나타난 본 발명의 첨가조성물을 10중량부 혼합한 배지에 줄기세포를 배양하고, 이를 상술한 과정을 따라 성장시키고, 유효인자를 추출하였다.Stem cells were cultured in a medium mixed with 10 parts by weight of the additive composition of the present invention shown in Table 1 in 100 parts by weight of DMEM (Dulbecco's Modified Eagle Medium), grown according to the procedure described above, and effective factors were extracted.
[ 실시예 5 ][Example 5]
DMEM(Dulbecco's Modified Eagle Medium) 100중량부에 표 1에 나타난 본 발명의 첨가조성물을 15중량부 혼합한 배지에 줄기세포를 배양하고, 이를 상술한 과정을 따라 성장시키고, 유효인자를 추출하였다.Stem cells were cultured in a medium mixed with 15 parts by weight of the additive composition of the present invention shown in Table 1 in 100 parts by weight of DMEM (Dulbecco's Modified Eagle Medium), grown according to the procedure described above, and effective factors were extracted.
[ 실시예 6 ][Example 6]
DMEM(Dulbecco's Modified Eagle Medium) 100중량부에 표 1에 나타난 본 발명의 첨가조성물을 20중량부 혼합한 배지에 줄기세포를 배양하고, 이를 상술한 과정을 따라 성장시키고, 유효인자를 추출하였다. Stem cells were cultured in a medium mixed with 20 parts by weight of the additive composition of the present invention shown in Table 1 in 100 parts by weight of DMEM (Dulbecco's Modified Eagle Medium), grown according to the above-described procedure, and effective factors were extracted.
[ 실시예 7 ][Example 7]
DMEM(Dulbecco's Modified Eagle Medium) 100중량부에 표 1에 나타난 본 발명의 첨가조성물을 25중량부 혼합한 배지에 줄기세포를 배양하고, 이를 상술한 과정을 따라 성장시키고, 유효인자를 추출하였다.Stem cells were cultured in a medium mixed with 25 parts by weight of the additive composition of the present invention shown in Table 1 in 100 parts by weight of DMEM (Dulbecco's Modified Eagle Medium), grown according to the procedure described above, and extracted with effective factors.
[ 실시예 8 ][Example 8]
DMEM(Dulbecco's Modified Eagle Medium) 100중량부에 표 1에 나타난 본 발명의 첨가조성물을 30중량부 혼합한 배지에 줄기세포를 배양하고, 이를 상술한 과정을 따라 성장시키고, 유효인자를 추출하였다.Stem cells were cultured in a medium mixed with 30 parts by weight of the additive composition of the present invention shown in Table 1 in 100 parts by weight of DMEM (Dulbecco's Modified Eagle Medium), grown according to the above-described procedure, and effective factors were extracted.
[ 실시예 9 ][Example 9]
DMEM(Dulbecco's Modified Eagle Medium) 100중량부에 표 1에 나타난 본 발명의 첨가조성물을 35중량부 혼합한 배지에 줄기세포를 배양하고, 이를 상술한 과정을 따라 성장시키고, 유효인자를 추출하였다.Stem cells were cultured in a medium mixed with 35 parts by weight of the additive composition of the present invention shown in Table 1 in 100 parts by weight of DMEM (Dulbecco's Modified Eagle Medium), grown according to the procedure described above, and extracted with effective factors.
[ 실시예 10 ][Example 10]
DMEM(Dulbecco's Modified Eagle Medium) 100중량부에 표 1에 나타난 본 발명의 첨가조성물을 40중량부 혼합한 배지에 줄기세포를 배양하고, 이를 상술한 과정을 따라 성장시키고, 유효인자를 추출하였다.Stem cells were cultured in a medium mixed with 40 parts by weight of the additive composition of the present invention shown in Table 1 in 100 parts by weight of DMEM (Dulbecco's Modified Eagle Medium), grown according to the procedure described above, and effective factors were extracted.
[ 실시예 11 ][Example 11]
DMEM(Dulbecco's Modified Eagle Medium) 100중량부에 표 1에 나타난 본 발명의 첨가조성물을 45중량부 혼합한 배지에 줄기세포를 배양하고, 이를 상술한 과정을 따라 성장시키고, 유효인자를 추출하였다.Stem cells were cultured in a medium mixed with 45 parts by weight of the additive composition of the present invention shown in Table 1 in 100 parts by weight of DMEM (Dulbecco's Modified Eagle Medium), grown according to the procedure described above, and extracted with effective factors.
[ 실시예 12 ][Example 12]
DMEM(Dulbecco's Modified Eagle Medium) 100중량부에 표 1에 나타난 본 발명의 첨가조성물을 50중량부 혼합한 배지에 줄기세포를 배양하고, 이를 상술한 과정을 따라 성장시키고, 유효인자를 추출하였다.Stem cells were cultured in a medium in which 50 parts by weight of the additive composition of the present invention shown in Table 1 was mixed with 100 parts by weight of DMEM (Dulbecco's Modified Eagle Medium), grown according to the above-described procedure, and effective factors were extracted.
다음으로, 본 발명에 의한 세포 배양용 배지 조성물에 의한 세포를 배양한 결과 세포가 수득된 양을 비교한 실험결과를 설명한다. Next, the experimental results comparing the amount obtained by culturing the cells with the cell culture medium composition according to the present invention will be described.
표 2에 나타난 바와 같이, 실시예 2 내지 실시예 12의 결과 세포의 수득량이 높음을 확인할 수 있다. 다만, 실시예 2의 경우 혈청을 투입하는 실시예 1과 근사하며, 실시예 11 및 실시예 12는 증가율이 상대적으로 낮아 수득 효율이 떨어지는 것을 확인할 수 있다. As shown in Table 2, it can be confirmed that the yield of cells was high as a result of Examples 2 to 12. However, in the case of Example 2, it is similar to Example 1 in which serum is added, and it can be confirmed that in Examples 11 and 12, the increase rate is relatively low and thus the yield efficiency is low.
다음으로, 본 발명에 의한 세포 배양용 배지 조성물에 의한 세포를 배양한 결과 수득된 세포를 파쇄한 결과 유효성분의 검출량을 비교한 실험결과를 설명한다. 특히, 실시예 1과 비교하여 첨가조성물의 추가량이 동일한 실시예 6을 비교하였다. Next, the experimental results comparing the detection amount of the active ingredient as a result of crushing the cells obtained by culturing the cells with the cell culture medium composition according to the present invention will be described. In particular, compared to Example 1, Example 6 in which the added amount of the added composition was the same was compared.
표 3에 나타난 바와 같이, 실시예 6의 결과 세포의 유효성분의 검출량이 높음을 확인할 수 있다. 또한, 실시예 1과 비교하여 수득량이 유사하거나 큰 실시예 3 내지 실시예 10의 경우 실시예 1 및 비교예 1에 비하여 검출량이 높을 것임을 예상할 수 있다. As shown in Table 3, the result of Example 6 can be confirmed that the detection amount of the active ingredient of the cell is high. In addition, it can be expected that the detection amount will be higher than in Example 1 and Comparative Example 1 in the case of Examples 3 to 10 in which the yield is similar or large compared to Example 1.
이하 줄기세포파쇄추출물이 연골세포에 작용하여 항 관절염 및 연골 재생 효과를 확인하는 시험을 상세하게 설명한다.Hereinafter, a test for confirming the anti-arthritis and cartilage regeneration effect by acting on the chondrocytes of the stem cell crushing extract will be described in detail.
1-1. 1-1. 관절연골세포의Articular cartilage 분리 및 배양 Isolation and culture
시험 전, Wistar rat 계 암컷 흰쥐의 연골을 재취하여 10% FBS가 포함된 DMEM 배지에서 배양 후, 연골세포의 특정마커인 CD44를 이용해 연골세포 임을 확인한다.(배양조건 37°5% CO2 )Before the test, the cartilage of Wistar rat-based female rats is recollected and cultured in DMEM medium containing 10% FBS, and then confirmed to be chondrocytes using CD44, a specific marker of chondrocytes. (Cultivation condition 37°5% CO 2 )
1-2. 결과1-2. result
연골세포의 특정마커인 CD44를 이용하여 연골세포임을 확인하였다. Chondrocytes were identified using CD44, a specific marker of chondrocytes.
2-1. 세포독성 및 2-1. Cytotoxicity and NO생성NO generation 억제효과 Inhibitory effect
1) 연골세포에 줄기세포파쇄성분추출물을 농도별 처리하여 24시간 배양, 세포생존율을 측정하여 물질의 세포에 대한 독성을 확인한다. 1) The chondrocyte is treated with stem cell crushing component extract by concentration, and cultured for 24 hours to measure cell viability to check the toxicity of the substance to the cells.
2) 연골세포에 IL-1α를 처리해 염증을 일으킨다.2) Treatment of IL-1α to chondrocytes causes inflammation.
3) 염증을 일으킨 연골세포에 줄기세포파쇄성분추출물을 농도별 처리하여 염증매개 인자인 NO생성 억제를 확인한다.(*NO;Nitric Oxide)3) Inflammation of the chondrocytes causing inflammation by treating the stem cell crushing component extract according to the concentration to confirm the inhibition of NO production as an inflammation mediator (*NO; Nitric Oxide)
2-2. 결과 2-2. result
1) 농도별 처리하여 세포생존율을 측정한 결과, 20 ng까지의 농도에서 세포독성을 나타내지 않았다. (농도 0,5,10,20 ng/ml)1) As a result of measuring cell viability by treatment by concentration, it did not show cytotoxicity at concentrations up to 20 ng. (
2) 염증유도물질인 IL-1α를 처리한 세포에서는 NO가 증가함 확인하였다. 염증을 유도한 연골세포에 줄기세포파쇄성분추출물을 처리한 결과 20ng/ml의 농도에서 약 30%의 염증억제 반응을 확인하였다.(농도 염증유도안한세포, 염증유도한세포, 5,10,20ng)2) NO was increased in cells treated with the inflammatory inducer IL-1α. As a result of treating the stem cell crushing component extract on the inflammation-induced chondrocytes, about 30% of the inflammation suppression reaction was confirmed at a concentration of 20 ng/ml. (Concentrated inflammation-inducing cells, inflammation-inducing cells, 5,10,20ng )
3-1. 3-1. iNOSiNOS , COX-2 및 , COX-2 and NFNF -- kBkB 발현 변화 Expression change
1) IL-1α 처리한 연골세포에 양성대조군인 Carprofen(비스테로이드성 물질)과 줄기세포파쇄성분추출물의 적용하여 NO생성과 COX-2, NF-kB 발현의 연관성을 확인한다. 1) Application of Carprofen (non-steroidal substance), a positive control, and stem cell crushing component extract to IL-1α-treated chondrocytes confirms the association between NO production and COX-2 and NF-kB expression.
NO: 염증이 발생하였을 때 증가하는 수치NO: increased value when inflammation occurs
COX-2 : 일종의 단백질, 염증인자에 의해 활성이 증가했다가 염증이 사라짐에 따라 감소하는 물질COX-2: a kind of protein, a substance that increases in activity by an inflammatory factor and then decreases as inflammation disappears
2) 염증을 일으킨 연골세포에 줄기세포파쇄성분추출물의 처리가 NF-κB의 발현에 미치는 영향을 확인한다.2) The effect of the treatment of the stem cell disruption component extract on the inflammatory cartilage cells on the expression of NF-κB is confirmed.
3-2. 결과3-2. result
1) IL-1α 처리로 유도된 연골세포에 줄기세포파쇄성분추출물을 처리한 결과1) Results of treating stem cell crushing component extract on chondrocytes induced by IL-1α treatment
IL-1α 처리로 유도된 NO의 생성 억제 효과는 iNOS와 COX-2의 발현 억제와 연관이 있음을 의미한다.The inhibitory effect of NO production induced by IL-1α treatment is related to the inhibition of expression of iNOS and COX-2.
2) 줄기세포파쇄성분추추물을 처리한 연골세포는 IL-1α에 의한 NF-κB 단백질이 핵내로 이동되는 것을 억제함으로써 IL-1α에 의해 유도되는 주요 염증 신호전달 경로인 NF-κB pathway를 차단 할 수 있음을 확인하였다.2) Chondrocytes treated with stem cell crushing component extracts block the NF-κB pathway, a major inflammatory signaling pathway induced by IL-1α by inhibiting NF-κB protein migration into the nucleus by IL-1α. It was confirmed that it can be done.
4. 4. iMAPKsiMAPKs 활성에 미치는 영향 Effect on activity
1) IL-1α 에 의해 염증이 발생한 연골세포에 줄기세포파쇄성분추출물을 처리하여 iMAPKs 활성을 확인한다.1) Stem cell disruption component extract is treated with chondrocytes inflamed by IL-1α Confirm iMAPKs activity.
1)항염증 기전확인1) Confirmation of anti-inflammatory mechanism
줄기세포파쇄성분추출물의 항염증 효능이 p38 MAPK 신호전달 억제를 통하여 이루어짐을 의미한다.It means that the anti-inflammatory effect of the stem cell crushing component extract is achieved through suppression of p38 MAPK signaling.
5-1. 연골형성 및 발달에 관여하는 인자 5-1. Factors involved in cartilage formation and development qPCRqPCR 측정 Measure
1) 유전학적 발현을 통해 본 줄기세포성분추출물의 항염즘 효과 확인한다.1) Confirm the anti-inflammatory effect of this stem cell component extract through genetic expression.
2）연골 형성 발달에 관여하는 sox-9의 유전자를 통해 연골형성을 확인한다.2) Check cartilage formation through the gene of sox-9, which is involved in cartilage formation development.
5-2. 결과5-2. result
1) MMPs는 연골조직의 이화작용에 관여하는 주요 단백질로서, IL-1α(염증유발물질)를 처리했을 때 MMP-13의 유전자 발현이 크게 증가되었으나, 줄기세포파쇄성분추출물의 처리 시 발현이 감소되는 것을 확인하였다.1) MMPs are major proteins involved in the catabolism of cartilage tissue, and when IL-1α (inflammation-inducing substance) was treated, gene expression of MMP-13 was significantly increased, but expression was decreased when the stem cell disruption component was treated. It was confirmed.
2) 연골 형성 및 발달에 관여하는 SOX-9의 유전자도 줄기세포파쇄성분추출물의 농도별 처리에 따라 유전자 발현이 증가되는 것을 확인하였다.2) The gene of SOX-9, which is involved in cartilage formation and development, was also confirmed to increase in gene expression according to the concentration-specific treatment of the stem cell disruption component extract.
이하 줄기세포파쇄추출물이 항염증 작용 효과를 확인하는 시험을 상세하게 설명한다.Hereinafter, a test for confirming the anti-inflammatory effect of the stem cell crushing extract will be described in detail.
1-1. 세포독성평가1-1. Cytotoxicity evaluation
1) 줄기세포파쇄추출물(T-stem)을 0.1㎍/ml부터 3㎍/ml의 농도로 마우스 대식세포 (RAW 246.7)에 24시간 동안 처리하였다. 1) Stem cell disruption extract (T-stem) was treated with mouse macrophages (RAW 246.7) at a concentration of 0.1 μg/ml to 3 μg/ml for 24 hours.
2) 줄기세포파쇄추출물과 LPS 동시에 처리하였다. 2) Stem cell disruption extract and LPS were treated simultaneously.
1-2. 결과 1-2. result
1) 도 10에 도시된 바와 같이, 줄기세포파쇄추출물 단독으로 농도별 처리결과, 통계적으로 유의한 세포 독성이 나타나지 않았다. 1) As shown in FIG. 10, as a result of treatment by concentration with the stem cell crushing extract alone, there was no statistically significant cytotoxicity.
2) 도 11에 도시된 바와 같이, 마우스 대식세포의 염증반응 유발물질인 Lipopolysaccharide (LPS) 1㎍/ml과 함께 줄기세포파쇄추출물을 처리한 결과 세포 독성이 나타나지 않았다.2) As shown in FIG. 11, as a result of treating the stem cell crushing extract with 1 μg/ml of Lipopolysaccharide (LPS), which is an inflammatory reaction inducer of mouse macrophages, cytotoxicity was not observed.
2-1. 세포형태변화2-1. Cell morphology change
줄기세포파쇄추출물을 2㎍/ml을 마우스 대식세포 (RAW 246.7)에 24시간 처리한 후, 세포형태 변화를 확인하였다. After the stem cell crushing extract was treated with 2 μg/ml of mouse macrophages (RAW 246.7) for 24 hours, changes in cell morphology were confirmed.
2-2. 결과2-2. result
염증유발인자인 LPS 1㎍/ml을 처리한 그룹에서 대조군 그룹에 비해 대식세포의 활성화가 비교적 증가함을 확인하였다.In the group treated with the
도 12에 도시된 바와 같이, 줄기세포파쇄추출물과 LPS 1㎍/ml을 함께 처리한 세포에서 LPS 1㎍/ml를 단독으로 처리한 그룹에 비해 대식세포의 활성화가 비교적 적게 발생하였다.As shown in FIG. 12, in cells treated with stem cell crushing extract and
3-1. 산화질소3-1. Nitric oxide
생성염증유발인자인 LPS 1㎍/ml를 전처리한 마우스 대식세포(RAW 246.7)에 줄기세포파쇄추출물을 0.4㎍/ml에서 2㎍/ml까지 농도로 24시간 처리한 후, griess assay를 통해 세포의 산화질소농도를 확인하였다. Stem cell disruption extracts were treated with 0.4 μg/ml to 2 μg/ml of stem macrophage extracts in mouse macrophages (RAW 246.7) pre-treated with 1 μg/ml of LPS, a productive inducer, and then subjected to gries assay. The nitrogen oxide concentration was confirmed.
3-2. 결과 3-2. result
LPS 자극에 의해 산화질소의 생성이 증가하였다. The production of nitric oxide was increased by LPS stimulation.
도 13에 도시된 바와 같이, LPS만 처리한 그룹에 비해 LPS와 줄기세포파쇄추출물을 같이 처리한 그룹에서 산화질소의 농도가 통계적으로 유의한 감소를 확인하였다. As shown in Figure 13, compared to the LPS-only group, the concentration of nitrogen oxide in the group treated with LPS and stem cell crushing extract was confirmed to be a statistically significant decrease.
4-1. 4-1. iNOSiNOS 및 COX-2 단백질 발현 변화 And COX-2 protein expression changes
염증유발인자인 LPS 1㎍/ml를 전처리한 마우스 대식세포(RAW 246.7)에 시험물질 줄기세포파쇄추출물을 1㎍/ml과 2㎍/ml의 농도로 24시간 처리한 후, western blot 분석을 통해 염증성 효소 iNOS와 COX2 단백질 발현을 확인하였다.After processing the test substance stem cell crushing extract at concentrations of 1 μg/ml and 2 μg/ml for mouse macrophages (RAW 246.7) pre-treated with 1 μg/ml of LPS, an inflammatory factor, through western blot analysis Expression of inflammatory enzymes iNOS and COX2 protein was confirmed.
4-2. 결과 4-2. result
도 14에 도시된 바와 같이, LPS만 처리한 그룹에 비해 시험물질 T-STEM을 같이 처리한 그룹에서 농도 의존적으로 iNOS와 COX-2 단백질 발현이 감소함을 확인하였다. As shown in FIG. 14, it was confirmed that iNOS and COX-2 protein expression decreased in a concentration-dependent manner in the group treated with the test substance T-STEM compared to the group treated with LPS only.
5-1. 염증성 사이토카인 생성5-1. Inflammatory cytokine production
염증유발인자인 LPS 1㎍/ml를 전처리한 마우스 대식세포(RAW 246.7)에 줄기세포파쇄추출물을 1㎍/ml과 2㎍/ml의 농도로 24시간 처리한 후, ELISA 분석법을 통해염증성 사이토카인 생성(TNFα, IL-1β)을 확인하였다. After treating the stem cell crushing extract with concentrations of 1 µg/ml and 2 µg/ml in mouse macrophages (RAW 246.7) pretreated with the
5-2. 결과 5-2. result
LPS 자극에 의해 염증성 사이토카인의 생성량이 증가함을 확인하였다. It was confirmed that the amount of inflammatory cytokines increased by LPS stimulation.
도 15 및 도 16에 도시된 바와 같이, 시험물질 줄기세포파쇄추출물 처리에 의해 염증성 사이토카인의 감소를 관찰하였다.As shown in Figures 15 and 16, a decrease in inflammatory cytokines was observed by treatment with the test material stem cell lysate extract.
본 발명에 의한 줄기세포 파쇄추출물의 유효성분의 효능은 다음과 같다. The efficacy of the active ingredient of the stem cell shredding extract according to the present invention is as follows.
먼저, 치매의 원인은 베타아밀로이드 독성단백질과 타우 단백질의 변성으로 발생되는데, 제대혈유래 중간엽 줄기세포를 알츠하이머병 생쥐의 양쪽 해마에 반복 이식했을 때 기억력 향상, 뇌 내 베타 아밀로이드의 양을 감소시키며, 생쥐의 뇌조직에서 베타 아밀로이드를 생성시키는 효소인 β -secretase1의 양이 감소되고, 미세아교세포의 염증성 시토카인의 분비가 억제되고 항염증 시토카인의 분비가 증가됨을 확인된다. 위 결과들로 인해 타우 단백질의 과인산화도 억제된다. First, the cause of dementia is caused by degeneration of beta amyloid toxic protein and tau protein. When umbilical cord blood-derived mesenchymal stem cells are repeatedly transplanted into both hippocampus of Alzheimer's disease mice, memory improves and reduces the amount of beta amyloid in the brain. It is confirmed that the amount of β-secretase1, an enzyme that generates beta amyloid in the brain tissue of mice, is reduced, the secretion of inflammatory cytokines of microglia is suppressed, and the secretion of anti-inflammatory cytokines is increased. Due to the above results, overphosphorylation of tau protein is also suppressed.
따라서, 중간엽 줄기세포가 알츠하이며 치매의 진행을 억제시키고, 치매의 원인이 되는 베타아밀로이드 독성단백질의 양을 감소시킨다는 결과가 있어, 줄기세포파쇄성분추출물(쉘드줄기세포)은 면역거부반응을 제거한 줄기세포 유효성분이 밀집되어있는 물질로 치매치료에 사용될 수 있을 것이다. 또한, 본 발명을 통해 세포 수득률, 유효성분의 수득률을 높였으므로, 기존 줄기세포 자체를 사용하는 것보다 위험성이 적고 효과가 뛰어난 치료제로 사용될 수 있다. Therefore, the mesenchymal stem cells are Alzheimer's and suppress the progression of dementia, and there is a result of reducing the amount of beta amyloid toxic protein that causes dementia, and the stem cell disruption component extract (shell stem cells) has an immune rejection reaction. The removed stem cell active ingredient is dense and may be used for the treatment of dementia. In addition, since the cell yield and the yield of the active ingredient are increased through the present invention, it can be used as a therapeutic agent with less risk and superior effect than using the existing stem cells themselves.
그리고, 잇몸병은 치아뿌리와 잇몸의 뼈, 잇몸 등에 염증이 생김으로 인해 발병되는데, 본 발명의 줄기세포의 파쇄추출물이 잇몸, 잇몸뼈를 재생할 수 있도록 줄기세포가 도움이 될 수 있다.And, the gum disease is caused by inflammation of the roots of the teeth, the bones of the gums, the gums, etc., and the stem cells can help the shredded extract of the stem cells of the present invention regenerate the gums, gum bones.
즉, 줄기세포 성장인자들이 염증억제, 뼈, 연골, 잇몸세포 재생, 상처치유재생 촉진 도움을 주므로 잇몸치료제로 사용될 수 있다, 특히, 프로콜라겐, 그물망처럼 생긴 콜라겐 네트워크가 세포와 세포를 서로 단단하게 접착시켜주며, 세포가 만들어지고 혈관, 뼈, 관절 등 인체의 많은 부분이 제 기능을 하도록 도와주는 환경 제공을 제공하므로 잇몸병치료제로 사용될 수 있다. 또한, 본 발명을 통해 세포 수득률, 유효성분의 수득률을 높였으므로, 기존 줄기세포 자체를 사용하는 것보다 위험성이 적고 효과가 뛰어난 치료제로 사용될 수 있다. In other words, stem cell growth factors can be used as a gum treatment agent because they help inhibit inflammation, promote bone regeneration, cartilage, gum cell regeneration, and wound healing. In particular, collagen networks, such as procollagen and reticulum, harden cells and cells together. It can be used as a gum disease treatment because it provides adhesion and provides an environment that helps cells make and functions in many parts of the body, such as blood vessels, bones, and joints. In addition, since the cell yield and the yield of the active ingredient are increased through the present invention, it can be used as a therapeutic agent with less risk and superior effect than using the existing stem cells themselves.
그리고, 관절 및 관절주위염은 노화, 부상 등 관절주위 염증으로 인해 발생되는데, 본 발명의 줄기세포 파쇄추출물이 염증억제, 뼈, 연골, 잇몸세포 재생, 상처치유재생 촉진 도움을 주므로 잇몸치료제로 사용될 수 있다. 프로콜라겐 그물망처럼 생긴 콜라겐 네트워크가 세포와 세포를 서로 단단하게 접착시켜주며, 세포가 만들어지고 혈관, 뼈, 관절 등 인체의 많은 부분이 제 기능을 하도록 도와주는 환경 제공을 제공하므로, 관절염 및 관절주위염의 치료제로 사용될 수 있다. 또한, 본 발명을 통해 세포 수득률, 유효성분의 수득률을 높였으므로, 기존 줄기세포 자체를 사용하는 것보다 위험성이 적고 효과가 뛰어난 치료제로 사용될 수 있다. And, joints and periarthritis are caused by inflammation around the joints, such as aging and injury, and the stem cell crushing extract of the present invention can be used as a gum treatment agent because it helps suppress inflammation, promote bone cell cartilage, gum cell regeneration, and wound healing regeneration. have. The collagen network, which looks like a procollagen mesh, tightly bonds cells and cells to each other, and provides an environment that helps cells make and functions in many parts of the human body, such as blood vessels, bones, and joints. It can be used as a therapeutic agent. In addition, since the cell yield and the yield of the active ingredient are increased through the present invention, it can be used as a therapeutic agent having less risk and superior effect than using the existing stem cells themselves.
그리고, 탈모의 원인은 환경적요인, 유전적요인 등 다양한데, 본 발명에 의한 줄기세포 파쇄추출물 중 VEGF 성장인자가 모낭성장에 도움을 주고, KGF 및 모근을 자극하여 모발 강화에 도움을 주므로 탈모의 치료제로 사용될 수 있다. 또한, 본 발명을 통해 세포 수득률, 유효성분의 수득률을 높였으므로, 기존 줄기세포 자체를 사용하는 것보다 위험성이 적고 효과가 뛰어난 치료제로 사용될 수 있다. In addition, the causes of hair loss are various such as environmental factors and genetic factors. Among the stem cell crushing extracts according to the present invention, VEGF growth factor helps follicle growth, and stimulates KGF and hair roots to help strengthen hair, thereby helping to strengthen hair. It can be used as a therapeutic agent. In addition, since the cell yield and the yield of the active ingredient are increased through the present invention, it can be used as a therapeutic agent with less risk and superior effect than using the existing stem cells themselves.
그리고, 피부노화의 원인은 환경적 요인, 유전적 요인 등 다양한데, 본 발명인 줄기세포 파쇄추출물 중 KGF, 새로운 세포생성(콜라겐)기능을 통해 주름방지, 회복, 피부보호, 젊은피부유지 및 UV자극방어하는데 도움을 주고, 그물망처럼 생긴 콜라겐 네트워크가 세포와 세포를 서로 단단하게 접착시켜주어, 피부탄력에 도움을 주므로 피부치료제로 사용할 수 있다. 또한, 본 발명을 통해 세포 수득률, 유효성분의 수득률을 높였으므로, 기존 줄기세포 자체를 사용하는 것보다 위험성이 적고 효과가 뛰어난 치료제로 사용될 수 있다. And, the causes of skin aging are various factors such as environmental factors and genetic factors. Anti-wrinkle, recovery, skin protection, maintenance of young skin, and UV stimulation defense through KGF, new cell generation (collagen) function among stem cell crushing extracts of the present invention It can be used as a skin treatment agent because it helps the skin, and the collagen network that looks like a mesh firmly adheres the cells and the cells to each other and helps the skin elasticity. In addition, since the cell yield and the yield of the active ingredient are increased through the present invention, it can be used as a therapeutic agent with less risk and superior effect than using the existing stem cells themselves.
이와 같이, 상술한 본 발명의 기술적 구성은 본 발명이 속하는 기술분야의 당업자가 본 발명의 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시 될 수 있다는 것을 이해할 수 있을 것이다.As described above, it will be understood that the technical configuration of the present invention described above can be implemented in other specific forms without changing the technical concept or essential features of the present invention by those skilled in the art to which the present invention pertains.
그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌것으로서 이해되어야 하고, 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위에 나타나며, 특허청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.Therefore, the above-described embodiments are to be understood in all respects as illustrative and not restrictive, and the scope of the present invention appears in the following claims rather than the above detailed description, meaning and scope of the claims, and equivalents thereof. It should be construed that any altered or modified form derived from the concept is included in the scope of the present invention.
10: 몸체부 12: 제 1용기부
14: 제 2용기부 16: 파쇄부
18: 밸브 20: 펌프10: body portion 12: first container portion
14: second container portion 16: crushing portion
18: valve 20: pump
Claims (9)
히알루론산; 및
첨가조성물로 구성되고,
상기 첨가조성물은,
글리신, 히스티딘, 이소류신, 메티오닌, 페닐알라닌, 프롤린, 히드록시프롤린, 세린, 트레오닌, 트립토판, 티로신, 발린, bFGF, EGF, VEGF, KGF, HGF, TGF, 비타민C, 비타민B1, 비타민B12, 비타민E, 셀레늄, 트랜스페린을 포함하여 구성되며,
상기 히알루론산은 10㎍/㎖의 농도로 포함되고,
상기 첨가조성물에서
상기 글리신은 1ng/㎖의 농도로 포함되고,
상기 히스티딘은 1ng/㎖의 농도로 포함되고,
상기 이소류신은 1ng/㎖의 농도로 포함되고,
상기 메티오닌은 1ng/㎖의 농도로 포함되고,
상기 페닐알라닌은 1ng/㎖의 농도로 포함되고,
상기 프롤린은 10ng/㎖의 농도로 포함되고,
상기 히드록시프롤린은 5ng/㎖의 농도로 포함되고,
상기 세린은 1ng/㎖의 농도로 포함되고,
상기 트레오닌은 1ng/㎖의 농도로 포함되고,
상기 트립토판은 1ng/㎖의 농도로 포함되고,
상기 티로신은 1ng/㎖의 농도로 포함되고,
상기 발린은 2ng/㎖의 농도로 포함되고,
상기 bFGF는 9㎍/㎖의 농도로 포함되고,
상기 EGF는 1.5㎍/㎖의 농도로 포함되고,
상기 VEGF는 1㎍/㎖의 농도로 포함되고,
상기 KGF는 1.2㎍/㎖의 농도로 포함되고,
상기 HGF는 0.5㎍/㎖의 농도로 포함되고,
상기 TGF는 0.5㎍/㎖의 농도로 포함되고,
상기 비타민C는2㎍/㎖의 농도로 포함되고,
상기 비타민B1는 0.5㎍/㎖의 농도로 포함되고,
상기 비타민B12는 3㎍/㎖의 농도로 포함되고,
상기 비타민E는 500㎍/㎖의 농도로 포함되고,
상기 셀레늄은 1.8㎍/㎖의 농도로 포함되고,
상기 트랜스페린은 12㎍/㎖의 농도로 포함되고,
상기 기본배지 100중량부에 대하여 상기 첨가조성물은 5 내지 40중량부의 비율로 혼합되는 것을 특징으로 하는 지방줄기세포 배양용 배지 조성물.Basic medium;
Hyaluronic acid; And
It consists of an additive composition,
The additive composition,
Glycine, histidine, isoleucine, methionine, phenylalanine, proline, hydroxyproline, serine, threonine, tryptophan, tyrosine, valine, bFGF, EGF, VEGF, KGF, HGF, TGF, vitamin C, vitamin B1, vitamin B12, vitamin E, Contains selenium, transferrin,
The hyaluronic acid is contained in a concentration of 10㎍ / ㎖,
In the additive composition
The glycine is contained in a concentration of 1ng / ㎖,
The histidine is contained in a concentration of 1ng / ㎖,
The isoleucine is contained in a concentration of 1ng / ㎖,
The methionine is contained in a concentration of 1ng / ㎖,
The phenylalanine is contained in a concentration of 1ng / ㎖,
The proline is contained in a concentration of 10ng / ㎖,
The hydroxyproline is contained in a concentration of 5ng / ㎖,
The serine is contained in a concentration of 1ng / ㎖,
The threonine is contained in a concentration of 1ng / ㎖,
The tryptophan is included in a concentration of 1ng / ㎖,
The tyrosine is contained in a concentration of 1ng / ㎖,
The valine is included at a concentration of 2ng/ml,
The bFGF is contained at a concentration of 9 μg/ml,
The EGF is included at a concentration of 1.5 μg/ml,
The VEGF is contained in a concentration of 1 μg/ml,
The KGF is included at a concentration of 1.2 μg/ml,
The HGF is included at a concentration of 0.5 μg/ml,
The TGF is included at a concentration of 0.5 μg/ml,
The vitamin C is contained in a concentration of 2㎍ / ㎖,
The vitamin B1 is contained in a concentration of 0.5㎍ / ㎖,
The vitamin B12 is contained in a concentration of 3㎍ / ㎖,
The vitamin E is contained in a concentration of 500㎍ / ㎖,
The selenium is contained in a concentration of 1.8㎍ / ㎖,
The transferrin is contained in a concentration of 12㎍ / ㎖,
The medium composition for adipose stem cell culture, characterized in that the additive composition is mixed in a proportion of 5 to 40 parts by weight based on 100 parts by weight of the basic medium.
제 1항의 배지조성물의 배지에 상기 추출된 줄기세포를 배양하는 제 2단계;
상기 줄기세포를 계대배양하는 제 3단계;
상기 배양된 줄기세포에서 세포를 획득하는 제 4단계;
상기 획득된 세포를 파쇄하는 제 5단계;
상기 파쇄된 물질을 여과하는 제 6단계; 및
상기 6단계 여과된 물질을 동결건조하여 보관 하거나 활용하는 7단계;
를 포함하여 구성되고,
상기 제 5단계에서,
내부에 공간이 형성되는 외부와 내부가 차단되는 몸체부와,
상기 몸체부 내부에 마련되고 상방이 개구되는 제 1용기부와,
상기 제 1용기부와 비교하여 상대적으로 작은 크기로 형성되며, 상기 제 1용기부 내에 마련되며 상방이 개구되어 내부에 줄기세포가 투입되는 제 2용기부와,
상기 제 2용기부 내부의 줄기세포를 파쇄하는 파쇄부와, 상기 몸체부 내부로 공기의 유동을 단속하는 밸브와, 상기 몸체부 내부에서 공기를 흡입하는 펌프를 포함하는 추출장치를 이용하고,
상기 줄기세포는 저장성 용액에 투입후 상기 제 2용기부에 투입하는 것을 특징으로 하는 줄기세포파쇄추출물 제조방법.A first step of extracting stem cells from adipocytes;
A second step of culturing the extracted stem cells in a medium of claim 1;
A third step of subculturing the stem cells;
A fourth step of obtaining cells from the cultured stem cells;
A fifth step of crushing the obtained cells;
A sixth step of filtering the crushed material; And
7th step of storing or utilizing the 6th step filtered material by lyophilization;
It comprises,
In the fifth step,
The body part that blocks the outside and the inside where the space is formed inside,
A first container part provided inside the body part and opened upward;
It is formed in a relatively small size compared to the first container portion, and is provided in the first container portion and the second container portion into which the stem cells are introduced into the upper opening,
Using a crushing unit for crushing the stem cells inside the second container portion, a valve that regulates the flow of air into the body portion, and an extraction device including a pump for sucking air inside the body portion,
The stem cells are introduced into the storage solution, and then the stem cell crushing extract manufacturing method characterized in that it is put into the second container.
A composition having an anti-arthritis inhibitory effect, comprising the stem cell crushing extract of claim 5.
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| KR1020180074243A KR102121602B1 (en) | 2018-06-27 | 2018-06-27 | Crushed Stem Cell Extract(Shelled Stem Cell) Manufacturing Method Using Mass Culture Medium Composition Method and Constituent 3-low Extracting Method and A Treating Composition for Anti-Inflammatory and A Treating Composition for Cell Regeneration |
| PCT/KR2018/007652 WO2020004698A1 (en) | 2018-06-27 | 2018-07-05 | Preparation method for cell culture medium composition, preparation method for stem cell lysate extract (shelled stem cell) using preparation method for cell culture medium composition and three-conditional extraction method of effective ingredient from stem cell, anti-arthritis therapeutic composition using same, anti-inflammatory therapeutic composition using same, and cell-regenerating therapeutic composition |
| US16/038,595 US11261431B2 (en) | 2018-06-27 | 2018-07-18 | Crushed stem cell extract (shelled stem cell) manufacturing method using mass culture medium composition method and constituent 3-low extracting method and a treating composition for anti-inflammatory and a treating composition for cell regeneration |
| CN201810840633.1A CN110643567A (en) | 2018-06-27 | 2018-07-27 | Method for preparing medium composition for cell culture |
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